Molecular cloning of dihydroflavonol 4-reductase gene from grape berry and preparation of an anti-DFR polyclonal antibody

Authors

  • Ping Zhang
  • Peng-Fei Wen
  • Si-Bao Wan
  • Wei Wang
  • Qiu-Hong Pan
  • Ji-Chen Zhan
  • Wei-Dong Huang

DOI:

https://doi.org/10.5073/vitis.2008.47.141-145

Keywords:

DFR, Escherichia coli expression, antibody, purification, grape berry

Abstract

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is a key enzyme of the flavonoid pathway, which synthesizes numerous secondary metabolites to determine the quality of grape berry and wine. The full-length dfr cDNA with 1014 bp was cloned from grape berry, and then introduced into an expressed plasmid pET-30a (+) vector at the EcoR I and Xho I restriction sites. With induction of the isopropyl-β-D-thiogalactoside (IPTG), the pET-dfr was highly expressed in Escherichia coli BL21 (DE3) pLysS cells. A fusion protein with the His-Tag was purified through Ni-NTA His Bind Resin and then used as the antigen to immunize a New Zealand rabbit. The resulting antiserum was further purified precipitated by 50 % saturated ammonium sulfate and DEAE-Sepharose FF chromatography to obtain the immunoglobulin G (IgG) fraction. The resulting polyclonal antibody was found capable of immuno-recognizing the DFR of the crude protein extracts from grape berry. This work undoubtedly provides the possibility for further studies on biological regulation of DFR activity in grape berry.

 

Downloads

Published

2015-04-09

Issue

Vol. 47 No. 3 (2008): Vitis

Section

Article

License

The content of VITIS is published under a Creative Commons Attribution 4.0 license. Any user is free to share and adapt (remix, transform, build upon) the content as long as the original publication is attributed (authors, title, year, journal, issue, pages) and any changes to the original are clearly labeled. We do not prohibit or charge a fee for reuse of published content. The use of general descriptive names, trade names, trademarks, and so forth in any publication herein, even if not specifically indicated, does not imply that these names are not protected by the relevant laws and regulations. The submitting author agrees to these terms on behalf of all co-authors when submitting a manuscript. Please be aware that this license cannot be revoked. All authors retain the copyright on their work and are able to enter into separate, additional contractual arrangements.